The present invention relates to transport proteins, and in particular to transport proteins based on VP22, homologues of VP22 or fragments thereof, to molecules and compositions including the transport proteins, and to methods of delivering these proteins and any associated molecules to a target population of cells, typically at high efficiency.
The product of the herpes simplex virus type 1 (HSV-1) UL49 gene, the structural protein VP22 (4), is a major component of the HSV tegument, a compartment of the virion located outside the capsid and inside the envelope and composed of at least 10 or more additional virus polypeptides (for a review see (6)). VP22 has a molecular weight of 32 k, is very basic and is modified by phosphorylation and nucleotidylation (1,4,8,9,11) in the infected cell.
Despite being one of the major tegument proteins within the virion, together with the well characterised transcription regulatory protein VP16, little is known about the function of VP22 during the virus replicative cycle. It is not yet known if it is an essential virus protein, but it is possible that, in a manner similar to VP16, VP22 has two roles to perform during infectionxe2x80x94initially as a functional protein during viral gene expression, and subsequently as a structural component of the virion during virus assembly. With regard to the former, some evidence exists to suggest that VP22 can bind specifically to HSV-1 DNA (2,8,10).
We recently demonstrated that there is a stable and specific interaction between VP16 and VP22, which has implications for the mechanisms of action of VP16 in assembly and transcriptional activation. During those studies, we found that when VP22 was in cells by itself it had an unusual pattern of behaviour. In the work described here, we extended these studies to investigate the cellular localisation in detail, and found that VP22 exhibits a highly unusual property in that it is efficiently transported from the cell in which it is originally expressed, and in which it displays cytoplasmic localisation, to adjacent cells within the monolayer, in which it is taken up into the nucleus. This pattern of behaviour has not been observed for any other of a range of proteins we have tested, eg VP16 and to our knowledge these properties and activities are unprecedented.
This unexpected property of VP22 was observed when, approximately 30 hours after introduction of either VP22 or VP16 into a cell monolayer, while VP16 could be detected on average in about 2-5% of the cells (as is conventional in such experiments), VP22 could be detected in nearly every cell of the monolayer. We further found specificity in VP22 intercellular transport and have demonstrated the involvement of a determinant at the C-terminal end of the protein. This comes from the result that a variant lacking the C-terminal 34 amino acids, while being expressed in a cytoplasmic location in initially expressing cells, was not transported to adjacent cells.
Protein secretion or export normally occur via specific pathways requiring well characterised signal sequences for sorting into the compartments and vesicles involved in export pathways for a review see (12). VP22 does not possess any conventional signal sequences and its route and mechanism of transport is highly unusual and is previously uncharacterised. Further studies on the determinants required within VP22 and of the physiological requirements within the cell should help clarify the pathway involved.
Thus, VP22, or more particularly the determinants involved in VP22 transport, could be transferred to other proteins to enable transport and efficient expression or uptake within a target cell population. Widespread utility and applications of this property can easily be envisaged.
Accordingly, the present invention is based on the above finding that it is possible to introduce into a first part of a target population of cells nucleic acid encoding a transport protein, and optionally with nucleic acid encoding proteins associated with the transport protein (eg. as fusion partners), to express the nucleic acid, optionally from tissue specific promoters, to produce the protein(s), after which the transport protein is exported from the cells, together with any associated protein(s), to be taken up by a second part of the target population of cells not directly producing the protein(s). Typically, the protein(s) are found to be taken up by the second part of the population of cells at high efficiency, and tend to localise in the nuclei of the second part of the population of cells. Thus, the combination of initial introduction and subsequent transport allows the transport protein and any associated proteins to be delivered at high efficiency to the target population of cells.
Accordingly, in one aspect, the present invention provides a protein that is capable of being exported from the cells in which it is expressed and is capable of being taken up in other cells, for example those not directly producing the protein. Preferably, the transport protein is associated with one or more other proteins whose delivery to the populations of cells is desired.
Further experiments have confirmed that VP22 is imported into cells when it is added as an extract to the extracellular medium. This confirms that it is not necessary for the VP22 to be expressed in at least a part of the population of cells for the observed intercellular transport to occur.
In one alternative aspect, the VP22 transport protein can be coupled to the associated molecules, eg covalently, or incorporated with associated molecules, and used in that form, as opposed to the use of an expression vector to produce the protein and/or the associated molecule. In particular, this could allow non-peptidyl molecules, such as nucleic acid, drugs or markers (in addition to or as alternatives to proteins) to be associated to the transport protein, and be taken up into a population of cells, without the need to express the VP22 and the associated molecule in at least a part of the population of cells to which delivery of the VP22 and/or the associated molecule is desired.
In a preferred embodiment, the present invention provides a transport protein which is:
(i) VP22 or an active portion thereof;
(ii) a fragment or homologue of VP22 including one or more of the determinants providing the transport property; or,
(iii) a fragment from the C-terminal 34 amino acids of VP22;
the transport protein being optionally associated with one or more other molecules whose transport to the target population of cells is desired.
In this invention, xe2x80x9can active portionxe2x80x9d means a peptide which is less than full length VP22, but which retains the property of being secreted by the cells producing it and of being taken up other cells.
In a further aspect, the present invention provides a composition comprising one or more of the above transport proteins, the proteins being optionally associated with one or more molecules whose delivery to a population of cells is desired.
In a further aspect, the present invention provides a method of transporting a desired molecule into a population of cells by exposing the cells to the desired molecule and the transport protein as set out above.
Thus, in this aspect, the invention provides a method which avoids the need initially to transfect the population of cells with nucleic acid encoding the transport protein and optionally the desired molecule. Thus, this can also allow the transport of molecules which are non-peptidyl, which could not be expressed in a cell, as the desired molecule and the transport protein can be added to the the medium surrounding the cells.
In some embodiments, the desired molecule can be coupled covalently to the transport protein and these entities exposed to the target population of cells. Alternatively, the desired molecule and the transport protein can be non-covalently associated, eg using lipid based vehicles incorporating a desired molecule such as nucleic acid and the transport protein.
Accordingly, in a further aspect, the present invention provides a method of preparing a composition including a desired molecule for transport to a target population of cells comprising covalently or non-covalently associating the desired molecule and transport protein.
In a further aspect, the present invention provides nucleic acid encoding the transport protein and (optionally) the associated molecules. For example, in embodiments in which the transport protein is expressed as a fusion construct, the nucleic acid can be provided which encodes the transport protein and its fusion partner(s).
In a further aspect, the present invention provides expression vectors incorporating nucleic acid encoding the transport protein or an active portion or fragment thereof, and optionally nucleic acid encoding the associated molecules.
In a further aspect, the present invention provides host cells transfected with the above expression vectors.
In a further aspect, the present invention provides a method of transporting a desired protein or peptide to a target population of cells, the method comprising:
(i) transfecting, infecting or otherwise introducing into a first part of a target population of cells with nucleic acid encoding a transport protein, and optionally with nucleic acid encoding proteins associated with the transport protein;
(ii) expressing the nucleic acid to produce the protein(s), after which the transport protein is exported from the cells, together with any other proteins associated with it, to be taken up by a second part of the target population of cells not directly producing the protein(s).
It will be appreciated that introduction into transfection of a first part of a target population of cells could be achieved by infecting with a recombinant virus.
In a further aspect, the present invention provides a method of transporting a desired molecule into a population of cells comprising:
(I) coupling the desired molecule to a transport protein to form a complex; and,
(ii) exposing the cells to the complex so that the cells can take up the complex.
In this method, the desired molecule, which may be non-peptidyl, can be coupled to, e.g. covalently or incorporated with, the transport protein.
In a further aspect, the present invention provides the above transport proteins and vectors for use in methods of transporting molecules, both in research methods and in methods of therapy. The present invention is particularly applicable in methods of targeting agents to populations of cells where targeting at high efficiency is required, eg in gene therapy techniques, the treatment of cancer, eg by the delivery of tumour suppressing agents to cells.